Fabry disease is the 2nd-most prevalent lysosomal storage disorder (LSD) in humans. It is X-linked and pan-ethnic with a frequency of about1:40,000 males. In Fabry disease the deficiency of the lysosomal enzyme alpha-galactosidase A (a-gal A) are mainly manifested in the vascular endothelium. These cells build up excessive lipids with terminal galactose residues (mainly ceramide trihexoside; CTH) leading to vessel occlusions. A major source of CTH in Fabry disease is from the hematopoietic system-specifically from the breakdown of RBCs by macrophages. Patients succumb to renal, cardiovascular, or cerebrovascular disease in mid life. Current treatment for the disorder is only palliative. Unlike many other LSDs, limited primary nervous system involvement is observed in Fabry disease and even 5 percent of normal enzyme activity may improve the clinical course. An a-gal A deficient mouse has been created that offers a model for studies on the pathophysiology and the development of therapeutic strategies. Fabry disease and this model offer compelling systems to develop and test methods for the improvement of gene therapy. A phenomenon called metabolic cooperativity exists wherein genetically corrected cells secrete the hydrolase-which can be taken up and used by bystander cells. This allows for correction of a lower number of cells to impact therapy. We have previously created retroviral vectors and corrected a variety of Fabry patient and a-gal A-deficient mouse cells. We have also demonstrated that metabolic cooperativity occurs in vivo. The Specific Aims of this application are designed to further this therapeutic approach in order to improve outcomes for Fabry disease-and possibly for other LSDs. Hypothesis 1: In the a-gal A-deficient mouse pre-selection of retrovirally transduced cells co-expressing a selectable marker and the therapeutic gene will improve metabolic cooperativity over that seen with non-enriched cells, as measured systemically by increased levels of a-gal A activity and decreased CTH levels. Hypothesis 2: Retroviral vectors (including novel recombinant lentiviruses) can be generated that encode marking or the therapeutic a-gal A gene that lead to the specific amplification of transduced cells relevant to Fabry disease upon the addition of selectively active co-factors both in vitro and in vivo. Hypothesis: Co-overexpression of the prosaposin gene, a protein co-factor for the a-gal A enzyme, leads to higher catalytic activity in genetically corrected cells than overexpression of a-gal A alone.